GDNF is a neurotrophic factor that supports the development and survival of peripheral sympathetic, parasympathetic, enteric and sensory neurons as well as midbrain dopamine neurons and motoneurons. In various animal models of Parkinson's disease (PD) GDNF can prevent the neurotoxin-induced death of dopamine neurons and can promote axonal sprouting leading to functional recovery. Two GDNF splice variants, called pre-(α)pro-GDNF (previously called GDNFα) and pre-(β)pro-GDNF (previously called GDNFβ), have been described (Suter-Crazzolara and Unsicker, Neuroreport, 5:2486-2488 (1994)). These splice variants are produced by alternative splicing of the GDNF mRNA.
Many secreted proteins, including neurotrophic factors, are synthesized in the forms of precursors, pre-pro-mature proteins. The pre-region, consisting of the ER signal peptide, is clipped off during translation by a signal peptidase, and the pro-mature protein is released into the lumen of the ER immediately after being synthesized. The proteolytic cleavage of the mature protein can occur either inside the cell or in the extracellular matrix, or both. The pro-mature protein can also remain uncleaved and have different function than the cleaved mature protein. For example, both mature brain-derived neurotrophic factor (BDNF) and pro-BDNF are secreted from neuronal cells. Mature BDNF binds to TrkB receptor inducing neuronal survival, differentiation and synaptic modulation, whereas pro-BDNF binds to p75NTR and sortilin receptors inducing apoptosis (to review, see Thomas and Davies, Curr. Biol., 15:262-264 (2005); Teng et al., J. Neurosci., 25:5455-5463 (2005)).
The GDNF splice variants contain an amino terminal signal sequence (pre-region) and a pro-sequence which is cleaved from the mature domain (Lin et al., Science, 260:1130-1132 (1993)) (FIG. 1). The pro-region of (β)pro-GDNF is 26 amino acids (aa) shorter than the pro-region of (α)pro-GDNF (Trupp et al., J. Cell Biol., 130:137-148 (1995)). The mature GDNF proteins produced by both of these splice variants are most likely identical. Mature GDNF consists of 134 amino acids (aa) and contains two putative N-glycosylation sites as well as seven conserved cysteines in the same relative spacing as the other members of the TGF-β protein family (Lin et al., Science, 260:1130-1132 (1993); Eigenbrot and Gerber, Nat. Struct. Biol., 4:435-438 (1997); Chang et al., Endocri. Rev., 23:787-823 (2002)) (FIG. 1). The biologically active mature GDNF dimer is formed by a covalent disulfide bond between the unpaired cysteines in the monomers (Eigenbrot and Gerber, Nat. Struct. Biol. 4:435-438 (1997)).
In the scientific text, the names GDNF mRNA and GDNF protein have been used for the full-length pre-(α)pro-GDNF mRNA and for the mature GDNF protein that is produced by proteolytic cleavage of the (α)pro-GDNF protein. This mature GDNF protein has been extensively studied, and in PubMed more than 2500 citations are available for GDNF. GDNF was identified based on its ability to increase neurite length, cell size, and the number of dopaminergic neurons as well as their high affinity dopamine uptake in culture (Lin et al., Science, 260:1130-1132 (1993)). GDNF is a potent factor for the protection of nigral dopaminergic neurons against their toxin-induced degeneration in animal models of PD and also in the treatment of patients with PD (reviewed in Airaksinen and Saarma, Nat. Rev. Neurosci. 3:383-394 (2002) and Bespalov and Saarma, Trends Pharmacol. Sci. 28:68-74 (2007)). In addition, GDNF has a therapeutic role in the treatment of animal models of amyotrophic lateral sclerosis (ALS), addiction, alcoholism and depression (reviewed in Bohn, Exp. Neurol., 190:263-275 (2004); Messer et al., Neuron, 26:247-257 (2000); He et al., J. Neurosci., 25:619-628 (2005); Angelucci et al., Int. J. Neuropsycho-pharmacol., 6:225-231 (2003)). GDNF has important roles also outside the nervous system. It acts as a morphogen in kidney development and regulates the differentiation of spermatogonia (reviewed in Sariola and Saarma, J. Cell Sci. 116:3855-3862 (2003)).
The (α)pro-GDNF protein is disclosed in, e.g., U.S. Pat. No. 6,362,319 and European Patent No. 0 610 254, and a truncated form of GDNF in U.S. Pat. No. 6,184,200 and European Patent No. 0 920 448. Clinical trials for the treatment of Parkinson's disease have been carried out using the mature GDNF protein. Preclinical studies gave promising results (Gill et al., Nat. Med., 9:589-595 (2003); Slevin et al., J. Neurosurg., 102:401 (2005)), but the outcome of the Phase I/II trial was found disappointing. It was reported that improvements in Parkinson's symptoms were not statistically significant, and that there were potential safety risks. Therefore, the clinical trials with mature GDNF protein were totally halted (Lang et al., Ann Neurol., 59:459-466 (2006)).
The existence of the pre-(β)pro-GDNF mRNA splice variant was first described in rat tissues in 1994 by Suter-Crazzolara and Unsicker (Neuroreport, 5:2486-2488), in mouse tissues in 1997 by Matsushita et al. (Gene, 203:149-157 (1997)) and in human tissues in 1998 by Grimm et al. (Hum. Mol. Genet., 12:1873-1886 (1998)). In addition to mRNA expression data, Trupp et al. (J. Cell Biol., 130:137-148 (1995)) showed that the secreted GDNF protein, encoded by the pre-(β)pro-GDNF cDNA, promoted robust survival, extensive neurite outgrowth and increased cell body size in E10 chick paravertebral sympathetic neurons.